不同造模方式诱导的糖尿病小...织中脂质异位沉积情况的研究_刘露.pdf

中国病理生理杂志 Chinese Journal of Pathophysiology 2023，39（2）：276-286杂志网址：http:/不同造模方式诱导的糖尿病小鼠肝肾组织中脂质异位沉积情况的研究*刘露，扈腊英，王贵芳，常雪冰，黄亚莉，宋铃榆，周宇霞，郭兵（贵州医科大学病理生理学教研室/贵州省常见慢性疾病发病机制及药物研究重点实验室，贵州 贵阳 550025）摘要 目的：观察比较高脂饮食（high-fat diet，HFD）、链脲佐菌素（streptozocin，STZ）联合 HFD（HFD/STZ）、单侧肾脏切除术（unilateral nephrectomy，UNx）+HFD/STZ和db/db小鼠4种糖尿病模型小鼠肝肾组织中脂质异位沉积情况。方法：选取8周龄C57BL/6小鼠，随机分为正常饮食（normal chow diet，NCD）组（n=10）、HFD组（n=10）、HFD/STZ组（n=10）和UNx+HFD/STZ组（n=12）。NCD组给予NCD饲养；HFD组、HFD/STZ组和UNx+HFD/STZ组给予HFD饲养，诱导小鼠出现肥胖和胰岛素抵抗，且HFD/STZ组和UNx+HFD/STZ组给予55 mg/kg STZ腹腔注射，后以HFD饲养至40周龄。选取10周龄db/db小鼠和野生型小鼠各8只，NCD饲养至40周龄。记录分析小鼠血糖和体重变化；收集血液样本测定甘油三酯（triglyceride，TG）含量；肝肾组织样本石蜡包埋后进行苏木精-伊红染色观察组织病理变化；免疫荧光染色和Western blot检测脂滴包被蛋白2的表达；油红O染色和尼罗红染色观察脂滴沉积情况。结果：HFD组小鼠体重显著升高（P0.05）；HFD/STZ组、Unx+HFD/STZ组及db/db组血糖水平和血清TG含量显著升高（P0.05），且肝肾组织中存在大量脂质异位沉积，均能诱导小鼠发生糖尿病肝肾脏损伤，其中Unx+HFD/STZ组最严重；HFD组肾脏未见明显脂质沉积，肝脏存在大量脂质沉积。结论：Unx+HFD/STZ组小鼠的肝肾脂质沉积最严重，但存在死亡率较高、小鼠状态差等缺点。单纯HFD饮食的小鼠是研究非酒精性脂肪性肝病合适的模型。HFD/STZ和db/db模型小鼠出现糖尿病肾脏脂毒性且呈现糖尿病肾病的肾脏病理改变，类似于2型糖尿病患者，适用于研究糖尿病及其并发症。关键词 糖尿病；脂质沉积；脂毒性；链脲佐菌素；单侧肾脏切除术中图分类号 R587.2；R363.2 文献标志码 A doi：10.3969/j.issn.1000-4718.2023.02.010Comparative study on lipid deposition in liver and kidney tissues of diabetic mice induced by different modeling methodsLIU Lu，HU Laying，WANG Guifang，CHANG Xuebing，HUANG Yali，SONG Lingyu，ZHOU Yuxia，GUO Bing（Department of pathophysiology，Guizhou Medical University/Guizhou Key Laboratory of Pathogenesis and Drug Research of Common Chronic Diseases，Guiyang 550025，China.E-mail：zhouyuxia_；）ABSTRACT AIM：To observe the lipid deposition in liver and kidney tissues of diabetic mice.METHODS：Diabetes mellitus models were induced by high-fat diet（HFD），streptozocin（STZ）combined with HFD（HFD/STZ），unilateral nephrectomy（Unx）+HFD/STZ and db/db mice.Eight-week-old C57BL/6 mice were randomly divided into normal chow diet（NCD）group（n=10），HFD group（n=10），HFD/STZ group（n=10），and Unx+HFD/STZ group（n=12）.The mice in NCD group were fed with NCD.The mice in HFD group，HFD/STZ group and Unx+HFD/STZ group were fed with HFD.The mice in HFD/STZ group and Unx+HFD/STZ group were injected with STZ（55 mg/kg）and then continuously fed with HFD until to the 40th week.Ten-week-old db/db and wild-type mice（n=8）were fed with NCD until to the 40th week.Blood glucose level，body weight and serum triglyceride（TG）level of each group were detected.Tissue sections of the liver and kidney were made via dehydration and paraffin embedding.After hematoxylin-eosin staining，the histopatho文章编号 1000-4718（2023）02-0276-11 收稿日期 2022-09-19 修回日期 2022-11-03*基金项目 国家自然科学基金资助项目（No.82000741；No.32160207）；贵州省科技厅计划项目（黔科合基础-ZK 2021 一般402号）；中国博士后科学基金资助项目（No.2020M683374）；贵州省教育厅青年科技人才成长项目（黔科KY字 2021 170号）；贵州医科大学优秀青年人才计划（No.2021105）通讯作者 周宇霞 Tel：18750238963；E-mail：zhouyuxia_；郭兵 Tel：13908518950；E-mail：276logical changes of the liver and kidney tissues were observed by microscopy.The expression of perilipin 2 was detected by immunofluorescence staining and Western blot.Lipid deposition was observed by oil red O staining and Nile red staining.RESULTS：In HFD group，the body weight of the mice was significantly increased（P0.05）.In HFD/STZ group，Unx+HFD/STZ group and db/db group，both blood glucose level and serum TG content were significantly increased（P0.05），lipid deposition and tissue damage were observed in the liver and kidney tissues.The lipid deposition in the liver and kidney tissues was the most obvious in Unx+HFD/STZ group.In HFD group，lipid deposition was observed in the liver but not in the kidney.CONCLUSION：In the liver and kidney tissues，the lipid deposition is the most obvious in Unx+HFD/STZ group，but there are some shortcomings such as high mortality and poor state of mice.The HFD model is an ideal model to study nonalcohol fatty liver disease.The HFD/STZ and db/db models are ideal models to study diabetes mellitus and its complications，which display lipotoxicity in the kidney，accompanied with the typical renal pathological changes of diabetic nephropathy，similar to the pathological change of type 2 diabetes mellitus patients.KEY WORDS diabetes mellitus；lipid deposition；lipotoxicity；streptozotocin；unilateral nephrectomy糖尿病（diabetes mellitus，DM）是一种慢性代谢性疾病，其特征是异常升高的血糖水平（高血糖症），在大多数情况下，DM 会导致外周组织如心血管系统、视网膜、神经系统、肝脏和肾脏等发生许多的并发症1。除了高血糖症之外，其他代谢因素如游离脂肪酸（free fatty acids，FFA）水平的增加、脂联蛋白（adiponectin）表达量的减少也会加快疾病的进程2。而脂质代谢紊乱可能导致大量脂质积累，一旦超过脂肪组织的储存能力，多余的脂质将沉积到非脂肪组织如心肌、胰腺、骨骼肌、肝脏和肾脏中，激活了生脂和生糖细胞信号通路，导致组织细胞功能障碍，正常生理过程遭到破坏，称为脂毒性（lipotoxicity）3-4。2型糖尿病（type 2 diabetes mellitus，T2DM）的发生发展与肥胖导致的脂肪因子水平改变有关，而肥胖和高脂血症是慢性肾病（chronic kidney diseases，CKD）最常见的独立危险因素，肾脏近端小管细胞更是脂质积聚的主要部位之一，这提示肾实质中的脂质积聚对肾功能有害2-3。肝脏是一种胰岛素敏感组织，可通过调节葡萄糖利用和糖异生之间的相互作用维持体内葡萄糖水平，在糖尿病及其并发症的病程中起着重要的作用，而胰岛素抵抗也通过多种机制影响肝脏脂质代谢，包括促进肝脏中的脂肪从头合成等5。研究表明，脂毒性可通过激活炎症、氧化应激、线粒体功能障碍和细胞死亡对肝、肾组织产生毒性作用6-7。根据国际糖尿病基金会（international diabetes foundation，IDF）的数据，预计到 2025年受 T2DM 影响的人数将增加到 3.8 亿8。超过 80%被诊断为T2DM 的人是肥胖的，而早期 DM 患者，通过降低10%的体重可使肥胖个体的临床糖尿病消失9。因此，研究T2DM的发病与脂质沉积的关系尤为重要。动物模型是科学研究中重要的工具，为了复制与人类病程最相似的疾病动物模型，为预防糖尿病及其并发症提供新策略，目前常选择小鼠作为实验动物，其生理结构与人类极为相似，且遗传资源非常丰富，造模方式包括单纯